Metalloproteinase and inhibitor expression profiling of resorbing cartilage reveals pro-collagenase activation as a critical step for collagenolysis

Abstract

Excess proteolysis of the extracellular matrix (ECM) of articular cartilage is a key characteristic of arthritis. The main enzymes involved belong to the metalloproteinase family, specifically the matrix metalloproteinases (MMPs) and a group of proteinases with a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS). Chondrocytes are the only cell type embedded in the cartilage ECM, and cell-matrix interactions can influence gene expression and cell behaviour. Thus, although the use of monolayer cultures can be informative, it is essential to study chondrocytes encapsulated within their native environment, cartilage, to fully assess cellular responses. The aim of this study was to profile the temporal gene expression of metalloproteinases and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), reversion-inducing cysteine-rich protein with Kazal motifs (RECK), and α2-macroglobulin (α2M), in actively resorbing cartilage. The addition of the pro-inflammatory cytokine combination of interleukin-1 (IL-1) + oncostatin M (OSM) to bovine nasal cartilage induces the synthesis and subsequent activation of pro-metalloproteinases, leading to cartilage resorption. We show that IL-1+OSM upregulated the expression of MMP-1, -2, -3, -9, 12, -13, -14, TIMP-1, and ADAMTS-4, -5, and -9. Differences in basal expression and the magnitude of induction were observed, whilst there was no significant modulation of TIMP-2, -3, RECK, or ADAMTS-15 gene expression. IL-1+OSM downregulated MMP-16,TIMP-4, and α2M expression. All IL-1+OSM-induced metalloproteinases showed marked upregulation early in the culture period, whilst inhibitor expression was reduced throughout the stimulation period such that metalloproteinase production would be in excess of inhibitors. Moreover, although pro-collagenases were upregulated and synthesized early (by day 5), collagenolysis became apparent later with the presence of active collagenases (day 10) when inhibitor levels were low. These findings indicate that the activation cascades for pro-collagenases are delayed relative to collagenase expression, further confirm the coordinated regulation of metalloproteinases in actively resorbing cartilage, and support the use of bovine nasal cartilage as a model system to study the mechanisms that promote cartilage degradation.